Diet-based Sodium Regulation in Sixth-Instar Grasshoppers, Schistocerca americana (Drury) (Orthoptera: Acrididae) Shelby Kerrin Kilpatrick and Spencer T. Behmer Texas A&M University, Department of Entomology Edited by Benjamin Rigby and Shelby Kerrin Kilpatrick Abstract: This study analyzed sodium intake by Schistocerca americana (Drury) (Orthoptera: Acrididae) grasshoppers using three different seedling wheatgrass based diet treatments to simulate a natural food source. Sodium is a key nutrient for grasshopper cells, nerves, and reproduction. Grasshoppers acquire sodium from plants that they consume. However, it is unclear if grasshoppers self-regulate their sodium intake. Additionally, if grasshoppers self-regulate their sodium intake, the extent to which they do is uncertain. Newly molted sixth-instar grasshoppers were fed one of three diets in which the level of sodium that they had access to was varied. The S. americana grasshoppers consumed significantly less of the 0.5 M added sodium only diet when presented with an option to choose between this diet and a no-sodium-added diet (t = 9.6026, df = 7, P < 0.0001). Grasshoppers in the 0.5 M added sodium only treatment consumed a significantly lower amount of food (P < 0.0001) and gained a significantly lower mean mass (P < 0.0001), compared to the grasshoppers in the no-sodium-added only treatment. Our results generally correlated with previous studies on Locusta migratoria (L.) (Orthoptera: Acrididae), and information about the ecological tolerances and nutritional requirements of grasshoppers. Our data suggests that S. americana grasshoppers are capable of self-regulating their sodium intake. Additionally, we show that high concentrations of sodium in grasshopper diets have a negative effect on body mass. Our study illustrates that diet-based sodium regulation is a factor in the relationship between insect herbivores and their environments. Keywords: physiology, nutrition, tolerance, salt, insect herbivory, 6th-instar For the majority of terrestrial plants, sodium important nutrient for grasshoppers and other (Na) is not required for growth, development, insects as well as plants (Clarkson and or reproduction (Maathuis 2014). Sodium Hanson 1980, Pedersen and Zachariassen can adversely affect plant communities by 2002, Boswell et al. 2008, Hasegawa 2013, reducing the amount of water, other nutrients, Pontes et al. 2017). The sodium ion, Na+, is and oxygen available to plants (Hasegawa essential for cell and nerve function in 2013). Gradients of sodium concentrations grasshoppers as well as egg production in occur on land as a result of magma flows, females (Boswell et al. 2008). ocean water levels, and human activity such as fertilizing and watering crops or salting Grasshoppers are primarily herbivorous roadways (Kaspari et al. 2014). Sodium is an insects and rely on plants for vital nutrients such as sodium. In one study, it was found similarly in this situation as they are also that grasshoppers grazed in areas fertilized orthopterans and require sodium in their diet with nitrogen and phosphorus more than in like most other insects. unfertilized areas, and even identified the areas of higher fertilization and migrated to In addition to behavioral aspects of sodium them within a single season (Sparks and regulation, the physiological effects of Cebrian 2015). Insects also modify their sodium on grasshoppers include body mass, behavior to regulate salt intake (Arms et al. development time, the concentration of 1974, Boggs and Jackson 1991, Chambers et sodium in tissue, and the concentration of al. 1997, Trumper and Simpson 1993, 1994; sodium in feces. However there has been Pontes et al. 2017). Depending on the little research on these specific effects related chemical complexity of the food it is applied to sodium on grasshopper physiology to and its concentration, sodium chloride can (Trumper and Simpson 1994, Simpson et al. either stimulate or deter feeding in 2006, Boswell et al. 2008). It has been grasshoppers (Trumper and Simpson 1994). reported that, when reared on a standardized Locust nymphs, Locusta migratoria (L.) diet, the concentration of sodium related to (Orthoptera: Acrididae), exhibit different body dry mass increases linearly as feeding patterns within hours of being fed grasshoppers age (Boswell et al. 2008). either a 2.48% Wesson’s salts by dry weight diet or a no-added-sodium diet, including The objectives of our study were to determine indications of increased foraging behavior in if S. americana grasshoppers self-regulate the locusts provided with the no-added- sodium intake as well as analyze how sodium diet (Trumper and Simpson 1994). In different concentrations of sodium in the diet a more extreme case of behavioral regulation affect S. americana body mass. We of sodium intake, Mormon crickets, hypothesized that the grasshoppers would Anabrus simplex Haldeman (Orthoptera: display evidence of sodium regulation based Tettigoniidae), forage for salt when marching on the previous studies described. We also and will even cannibalize each other to obtain expected that body mass in S. americana this limiting nutrient (Simpson et al. 2006). would be reduced when fed a diet with a high concentration of sodium. Animals, including insect herbivores, are known for selecting foods based on nutrient Materials and Methods intake targets to support their biological processes (Behmer 2009). In one study, A. Sodium intake was measured between three simplex preferred a 0.25 M solution of groups of eight sixth-instar S. americana sodium chloride over other offered grasshoppers by providing each group of concentrations ranging from 0.0 M to 2.0 M them with a different variation of a seedling (Simpson et al. 2006). Additionally, when L. wheatgrass based treatment, to mimic what a migratoria nymphs are presented with two grasshopper might eat in a natural different food sources, a diet with no-added- environment. Three different treatments were sodium and a diet with added salt above their used: no-added-sodium only (the control optimal level, they would be expected to eat treatment), 0.5 M added sodium only, or a amounts of both diets to reach a level of block of each of the two prepared diets. The sodium intake close to or at their level of amounts of food consumed and mass gained requirement (Simpson 1994). Schistocerca by the grasshoppers in each treatment were americana would be expected to behave analyzed. Study insects. The S. Americana blades at their bases. The grass was wrapped grasshoppers used in this study were reared in paper towels and placed in a FreeZone Plus in a colony setting at the Behmer Lab of 6 Liter Cascade Console Freeze Dry System Insect Physiology and Behavior at Texas (Labconco, Kansas City, MO) freeze dryer A&M University, College Station, Texas, until the wheatgrass was completely dry or up USA. Individual sixth-instar nymphs were to about a week. The freeze-dried seedling arbitrarily chosen from groups that were wheatgrass was ground into a fine powder newly molted within 24 h. The grasshoppers with a fine sieve fitted in an MF 10 basic were released back into the colony at the Microfine grinder drive (IKA Werke, conclusion of the experiment. Wilmington, NC) and stored in a -40°C freezer until it was needed. Diets. Two different diets were prepared, a diet without added sodium to represent a Diet preparation. The no-added-sodium diet standard wheatgrass based diet and a diet was prepared by combining 1 g of freeze- with 0.5 M added sodium. The main dried seedling wheatgrass powder component of each diet was freeze dried (Wilmington, NC) with 10 mL of distilled seedling wheatgrass which contains 0.5% water (H2O) and 100 mg of melted agar sodium by dry weight (S.T.B., unpublished (Sigma-Aldrich, Inc., Saint Louis, MO). The data). solution was poured into a 100 mm by 15 mm plastic petri dish (VWR, Radnor, PA) for Freeze-dried seedling wheatgrass solidification. This resulted in a final diet that preparation. Wheatgrass seeds were planted was 8.79% sodium by dry weight. Blocks of in an equal mixture of potting soil and diet ranging from about 400 mg and 1300 mg vermiculite in a germination tray and covered in weight were cut out of the petri dishes and until they sprouted. Once sprouted, the wheat weighed to the nearest 0.10 mg using an grass was transferred to the Norman Borlaug electronic bench scale immediately before Institute for International Agriculture, Texas being presented to grasshoppers (Fig. 1). A&M University, College Station, Texas, USA, greenhouse and grown until they were Linear regression equations. A linear between 15-20 cm tall. During this time, the regression model was created to convert the wheatgrass was watered as needed to prevent wet mass of each diet to its final dry mass desiccation and promote growth. The wheat using Microsoft® Excel for Mac, version grass was harvested by cutting the grass 2013 (Microsoft, Redmond, WA). The data Fig. 1. Food dishes, prepared with blocks of diet, prior to being presented to the grasshoppers during the experiment. for the linear regression model was obtained respective diet presented to the grasshoppers by completely drying 12 blocks of each diet throughout the study so that the mass of food ranging between 400 mg and 1300 mg in consumed by the grasshoppers could be weight in a Model 20 Gravity Convection calculated. (GC) Lab Oven (Quincy Lab Inc., Chicago, IL) set at 40°C. The no-added-sodium diet Study design. Arenas were created with clear linear regression equation was plastic chambers measuring 19 cm long by 14 y = 0.0998x – 0.6103 (R² = 0.99828) (Fig. 2). cm wide by 9 cm high with six, 5 mm air The 0.5 M added sodium diet linear holes evenly spaced in the lid (Fig. 4). A wire regression equation was y = 0.1345x – 0.1420 mesh roost measuring 17 cm long (bent (R² = 0.99794) (Fig.